The recent growth in many areas of biotechnology has increased the demand to perform a variety of studies, commonly referred to as assays, of biochemical systems. These assays include for example, biochemical reaction kinetics, DNA melting point determinations, DNA spectral shifts, DNA and protein concentration measurements, excitation/emission of fluorescent probes, enzyme activities, enzyme co-factor assays, homogeneous assays, drug metabolite assays, drug concentration assays, dispensing confirmation, volume confirmation, solvent concentration, and solvation concentration. Also, there are a number of assays which use intact living cells that require visual examination.
Assays of biochemical systems are carried out on a large scale in both industry and academia, so it is desirable to have an apparatus that allows these assays to be performed in a convenient and inexpensive fashion. Because they are relatively easy to handle, are low in cost, and generally disposable after a single use, multiwell plates are often used for such studies. Multiwell plates are typically formed from a polymeric material and consist of an ordered array of individual wells. Each well includes sidewalls and a bottom so that an aliquot of sample can be placed within each well. The wells may be arranged in a matrix of mutually perpendicular rows and columns. Common sizes for multiwell plates include matrices having dimensions of 8×12 (96 wells), 16×24 (384 wells), and 32×48 (1536 wells).
The materials used to construct a multiwell plate are selected based on the samples to be assayed and the analytical techniques to be used. For example, the materials of which the multiwell plate is made should be chemically inert to the components of the sample or any biological or chemical coating that has been applied to the multiwell plate. Further, the materials should be impervious to radiation or heating conditions to which the multiwell plate is exposed during the course of an experiment and should possess a sufficient rigidity for the application at hand.
In many applications, a transparent window in the bottom of each well is needed. Transparent bottoms are primarily used in assay techniques that rely on emission of light from a sample within the well and subsequent spectroscopic measurements. Examples of such techniques include liquid scintillation counting and techniques which measure light emitted by luminescent labels, such as bioluminescent or chemiluminescent labels, fluorescent labels, or absorbance labels. Optically transparent bottom wells also enable microscopic viewing of specimens and living cells within the well. Currently, optically transparent and ultraviolet transparent bottomed multiwell plates exist in the market and are used for the aforementioned purposes. These multiwell plates are typically made from a hybrid of different polymeric materials, one material making up the sidewalls of the wells and another material making up the bottom walls of the wells.
In other applications, detection of the binding of unlabeled molecular species is accomplished by determining changes in the overall refractive index of an optical system that employs a biosensor in the bottom of each well of the microplate. Typically, an optical waveguide grating (OWG) sensor comprises a substrate, a grating, a dielectric layer deposited on the grating and a biologically active region on the dielectric layer. When spectral light is launched into such a system, detecting changes in the wavelength of light that is reflected back (resonant wavelength) provides information on the interactions that are occurring in the biological layer. Likewise, should a single wavelength of light be directed into the system at a particular angle of incidence, changes in the angle that the light is reflected from the structure provides information on interactions occurring in the biological layer. The utility of assays performed in such systems relies on making successive analytical observations interplayed between steps in the assay. This way, a true “before and after” analysis may be accomplished revealing the occurrence (or absence) of biological or chemical molecular interactions. Therefore, the repeatable and consistent alignment and/or positioning of a microplate incorporated into or onto a stage for analytical interrogation has been a requirement for these systems.
Current measurement protocol requires five primary steps: (1) initial/background measurement, (2) removal of the plate (for additional assay steps), (3) reinsertion of the plate into the reader, (4) second measurement, and (5) comparison of first and second measurements. Following the placement of a microplate into an exact location, an initial measurement can be read by a photometric/optical instrument. Once the microplate is removed, and manipulation of its contents completed, examination of the microplate depends on the exact repositioning of the microplate into the reader. Therefore, the second/final measurement result can be adversely affected by the slightest change, rotational and/or translational, in microplate position between the initial and second/final measurement steps. Small, lateral displacements (e. g. ˜1 micron) of the microplate (such as occur when the plate is removed and replaced) result in a spurious shift in the resonance wavelength. This arises since the small launch beam does not strike the same region of the grating during the initial measurement as it does during the final measurement and the fact that the grating resonance varies slightly with position. Accordingly, there is a need for a microplate design and method of analysis that will provide high levels of sensitivity while still allowing for slight lateral displacements within a detection system upon successive insertions into a reader. This need and other needs are satisfied by the multiwell plate and the method of the present invention.